We are studying the trafficking of HIV-1 macromolecules and assembly. Once it has exited the nucleus, HIV-1 RNA needs to travel to various subcellular locations to carry out its functions, including dimerizing with another viral RNA and assembling into a viral particle. Our current and future studies are focused on exploring the initiation of Gag:RNA interaction in the cells, examining RNA trafficking in T cells, and defining the role of the viral RNA genome in particle assembly. We will also determine the kinetics of virus maturation by live-cell imaging and determine the factors that shape RNA structures in the virions. _BACKGROUND: To generate infectious particles, HIV-1 RNA and proteins traffic to the plasma membrane, the major virus assembly site. The Gag protein drives HIV-1 assembly and interacts with viral RNA and proteins to ensure the packaging of the viral genome and replication machinery. Additionally, Gag interacts with host proteins for virus egress. It has often been suggested that the interactions of HIV-1 RNA and Gag leading to assembly are initiated in the cytoplasm. To better understand the regulation of virus assembly, we are examining cytoplasmic HIV-1 Gag:RNA and RNA:RNA interactions. We are also studying HIV-1 RNA trafficking in T cells and exploring the role of the RNA genome in HIV assembly. _Immature particles need to go through a maturation process to become infectious viruses. During this process, protease (PR) in the Gag-Pol polyprotein is activated and cleaves Gag/Gag-Pol polyproteins to release mature proteins. This process allows the rearrangement of the virion structure including the capsid proteins, which form a conical core. We are studying the timing of the proteolytic cleavage and virus maturation by using live-cell imaging. _The studies in this project seek to address several unanswered questions on the trafficking of HIV-1 macromolecules and virus assembly, which are essential processes in viral replication. _ACCOMPLISHMENTS: HIV-1 RNA must go to specific subcellular compartments to be translated or packaged into viral particles. Proper RNA trafficking is required for the functions of RNA and its encoded proteins. However, little was known about how HIV-1 RNA is transported in the cytoplasm. We visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules moved in a nondirectional, random-walk manner, and that the mean-squared distance traveled by the RNA increased linearly with time, indicative of diffusive movement. When Gag was expressed, a significant portion of HIV-1 RNA may be transported as Gag-RNA complexes, whose properties could differ greatly from Gag-free RNA. Therefore, we also analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to that of RNAs unable to express functional Gag. These studies have defined a major mechanism important to HIV-1 gene expression. Polarized T cells not only constitute a majority of HIV-1 target cells in vivo but also play a critical role in the spread of HIV-1 via cell-to-cell infection. To determine the distribution of HIV-1 RNA in polarized T cells, we visualized the RNA by using live-cell microscopy and a Bgl-YFP construct that specifically recognizes stem-loop sequences engineered into the HIV-1 genome. We found that HIV-1 RNAs were enriched near the uropod plasma membrane in a Gag-dependent manner. These results indicated that HIV-1 RNA is enriched during the process of virus assembly. As the Gag-enriched uropod is more likely to form a virological synapse, such targeting facilitates cell-mediated infection and virus spread in vivo. To better understand the trafficking of HIV-1 macromolecules, we are currently determining whether HIV-1 RNA and Gag can affect each other's subcellular localization and, if so, which elements are required for such effects. _To gain insights into RNA packaging and virus assembly mechanisms, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by total internal reflection fluorescence (TIRF) microscopy. HIV-1 RNA was labeled with a photo-convertible Eos protein via a BglG protein that recognizes stem-loop sequences engineered into the viral genome. UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. The dynamics of HIV-1 RNA were determined by photoconverting Eos near the plasma membrane and by following the population of the photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently. The presence of Gag significantly increased the time RNAs stay near the plasma membrane. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that was packaged into assembling viral complexes. We observed that the frequency of HIV-1 RNA packaging was dependent on the Gag expression level. Our results showed that only a small proportion of the HIV-1 RNAs (approximately one tenth to one third) that reached the plasma membrane was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained the temporal information of RNA-Gag interactions that lead to RNA encapsidation. We are currently studying whether HIV-1 RNA and Gag interact in the cytoplasm, and if so, what the biological consequences of such interactions are. __We have studied the role of HIV-1 RNA during virus assembly. It has been shown that in the absence of the viral RNA, HIV-1 particles contain cellular RNAs; thus, viral RNA is not required to form HIV-1 particles. We hypothesize that HIV-1 full-length RNA facilitates the formation of viral particles. To test our hypothesis, we examined the efficiencies of particle formation with and without RNA containing HIV-1 packaging signal. We found that, although viral particles can be generated without the presence of RNA genome, HIV-1 RNA genome facilitates the production of HIV-1 particles. Furthermore, the effects of RNA genome are dependent on the level of Gag expressed in the cells. These observations are consistent with our hypothesis that packaging a dimeric RNA is the nucleation process of HIV-1 assembly. We are currently dissecting the Gag properties required for RNA packaging.